ANG II-induced neointimal growth is mediated via cPLA2- and PLD2-activated Akt in balloon-injured rat carotid artery

Angiotensin II (ANG II) promotes neointimal growth in the balloon-injured rat carotid artery. However, the mechanism by which ANG II stimulates neointimal growth during vascular injury is not known. In cultured vascular smooth muscle cells, ANG II activates Akt through cytosolic phospholipase A2 (cPLA2)-dependent phospholipase D2 (PLD2). This study was conducted to determine whether ANG II-induced neointimal thickening is mediated via cPLA2- and PLD2-activated Akt in balloon-injured rat carotid arteries. ANG II-stimulated neointimal growth was inhibited by exposure of the injured carotid arteries to an adenovirus containing a dominant negative Akt mutant (intima-to-media ratio from 3.01 +/- 0.31 to 1.44 +/- 0.14, P < 0.01) or a retrovirus containing cPLA2 small interfering RNA (siRNA; intima-to-media ratio from 3.01 +/- 0.31 to 1.16 +/- 0.36, P < 0.001) or PLD2 siRNA (intima-to-media ratio from 3.01 +/- 0.31 to 1.33 +/- 0.11, P < 0.001). The effect of cPLA2 and PLD2 siRNA to reduce the ANG II-induced increase in neointimal thickening was associated with reduced expression of cPLA2 and PLD2 as determined by immunohistochemical analysis in injured carotid arteries. Western blot analysis showed that Akt phosphorylation that was increased by ANG II was inhibited in injured carotid arteries 2 days after exposure to cPLA2 or PLD2 siRNA or in injured arteries isolated after exposure to these agents for 30 min and then placed in tissue culture media for 24 h in the presence of these agents. These data suggest that the ANG II-induced neointimal growth is mediated by the activation of Akt through a mechanism dependent on cPLA2 and PLD2 activation in balloon-injured rat carotid arteries.     

Science, Stewardship, and Spirituality: The Human Body as a Model for Ecological Restoration

Ecological restoration is practiced by people of diverse backgrounds working at many different levels in a variety of settings. Portraying the human body as a metaphor of a natural ecosystem can be useful in identifying the breadth of strategies used to restore the natural environment. A technical approach, environmental stewardship, and more spiritual traditional attitudes to ecological restoration can be viewed as analogous to restoring health to the human body through an operation in a hospital, administering remedies to oneself from the medicine cabinet, and taking a broader holistic/preventative approach to cultivate the mind–body connection through, e.g., yoga.     

The role of chromatic and achromatic signals for fruit detection by birds

Fruit color changes during ripening are typically viewed asan adaptation to increase signal efficacy to seed dispersers.Plants can increase signal efficacy by enhancing chromatic (wavelengthrelated) and/or achromatic (intensity related) contrasts betweenfruit and background. To assess how these contrasts determinethe detectability of fruit signals, we conducted 2 experimentswith free-flying crows (Corvus ossifragus) under seminaturalconditions in a 2025 m² aviary. Crows searched first for artificialred and black fruits and detected red fruits from a larger distance.Because artificial red fruits had higher chromatic and lowerachromatic contrasts against foliage than artificial black fruits,crows apparently prioritized chromatic contrasts. Thus, thecommon change in fruit color from red to black during ripeningdoes not increase signal efficacy to crows. In a second trial,crows searched for UV-reflecting and black blueberries (Vaccinummyrtillus) against backgrounds of foliage and sand. Againstfoliage, UV-reflecting berries had higher chromatic and achromaticcontrasts than black berries, and crows detected them from alarger distance. Against sand, UV-reflecting berries had lowachromatic contrasts and black berries low chromatic contrasts.Crows detected both fruit types equally, suggesting that theyused chromatic contrasts to detect UV-reflecting berries andachromatic contrasts to detect black berries. Birds prioritizedchromatic contrasts when searching for artificial red fruitsin foliage but not when searching for blueberries on sand. Wesuggest that the relative importance of chromatic and achromaticcontrasts is contingent on the chromatic and achromatic varianceof the background. Models of signal perception can be improvedby incorporating background-specific effects.     

Effects of Prechilling and Sequential Washing on Enumeration of Microorganisms from Refuse

Techniques were evaluated for formation of a liquid inoculum from shredded municipal refuse, including chilling the refuse at 4°C prior to blending and multiple washing and blending cycles. The average count of cellulolytic bacteria from six different detachment treatments was 5.1 × 10⁴ cells per g (dry weight) of refuse with a range of 0.7 × 10⁴ to 12.7 × 10⁴ cells per g (dry weight). The liquid obtained from blending the refuse in phosphate buffer followed by hand squeezing was the selected detachment procedure. The inoculum formation procedure was validated by the addition of ruminal cellulolytic bacteria to refuse and recovery of the cellulolytic bacteria by most-probable-number enumerations. The ratio of measured to expected cell counts among tests in which different volumes of ruminal fluid were added to refuse ranged from 2.7 to 14.4. There was no evidence of anaerobic cellulolytic fungi in a refuse sample.     

Disruption of PTPRO causes childhood-onset nephrotic syndrome

Idiopathic nephrotic syndrome (INS) is a genetically heterogeneous group of disorders characterized by proteinuria, hypoalbuminemia, and edema. Because it typically results in end-stage kidney disease, the steroid-resistant subtype (SRNS) of INS is especially important when it occurs in children. The present study included 29 affected and 22 normal individuals from 17 SRNS families; genome-wide analysis was performed with Affymetrix 250K SNP arrays followed by homozygosity mapping. A large homozygous stretch on chromosomal region 12p12 was identified in one consanguineous family with two affected siblings. Direct sequencing of protein tyrosine phosphatase receptor type O (PTPRO; also known as glomerular epithelial protein-1 [GLEPP1]) showed homozygous c.2627+1G>T donor splice-site mutation. This mutation causes skipping of the evolutionarily conserved exon 16 (p.Glu854_Trp876del) at the RNA level. Immunohistochemistry with GLEPP1 antibody showed a similar staining pattern in the podocytes of the diseased and control kidney tissues. We used a highly polymorphic intragenic DNA marker-D12S1303-to search for homozygosity in 120 Turkish and 13 non-Turkish individuals in the PodoNet registry. This analysis yielded 17 candidate families, and a distinct homozygous c.2745+1G>A donor splice-site mutation in PTPRO was further identified via DNA sequencing in a second Turkish family. This mutation causes skipping of exon 19, and this introduces a premature stop codon at the very beginning of exon 20 (p.Asn888Lysfs*3) and causes degradation of mRNA via nonsense-mediated decay. Immunohistochemical analysis showed complete absence of immunoreactive PTPRO. Ultrastructural alterations, such as diffuse foot process fusion and extensive microvillus transformation of podocytes, were observed via electron microscopy in both families. The present study introduces mutations in PTPRO as another cause of autosomal-recessive nephrotic syndrome.     

Transduction of peptides and proteins into live cells by cell penetrating peptides

Internalization of peptides and proteins into live cells is an essential prerequisite for studies on intracellular signal pathways, for treatment of certain microbial diseases and for signal transduction therapy, especially for cancer treatment. Cell penetrating peptides (CPPs) facilitate the transport of cargo-proteins through the cell membrane into live cells. CPPs which allow formation of non-covalent complexes with the cargo are used primarily in this study due to the relatively easy handling procedure. Efficiency of the protein uptake is estimated qualitatively by fluorescence microscopy and quantitatively by SDS-PAGE. Using the CPP cocktail JBS-Proteoducin, the intracellular concentrations of a secondary antibody and bovine serum albumin can reach the micromolar range. Internalization of antibodies allows mediation of intracellular pathways including knock down of signal transduction. The high specificity and affinity of antibodies makes them potentially more powerful than siRNA. Thus, CPPs represent a significant new possibility to study signal transduction processes in competition or in comparison to the commonly used other techniques. To estimate the highest attainable intracellular concentrations of cargo proteins, the CPPs are tested for cytotoxicity. Cell viability and membrane integrity relative to concentration of CPPs are investigated. Viability as estimated by the reductive activity of mitochondria (MTT-test) is more sensitive to higher concentrations of CPPs versus membrane integrity, as measured by the release of dead cell protease. Distinct differences in uptake efficiency and cytotoxic effects are found using six different CPPs and six different adhesion and suspension cell lines.     

Apolipoprotein A-I isoforms in human lymph: effect of fat absorption

The effect of fat feeding (100 g of cream) on the apoA-I isoproteins distribution has been analyzed by two-dimensional gel electrophoresis in the chylomicrons, VLDL, LDL, and HDL isolated from the thoracic duct lymph of patients undergoing lymph drainage for immunosuppression, Isoforms apoA-I3 and apoA-I4 are the most abundant apoA-I isoproteins in plasma lipoproteins as well as in lymph lipoproteins collected in the fasting state. Fat feeding, on the other hand, results in a marked change in the apoA-I isoform pattern in lymph chylomicrons and VLDL, with a significant increase in the relative concentration of the apoA-I1 isoform. As a result the total concentration of this isoprotein in the lymph increased. The data indicate that fat feeding is associated with major changes in the distribution of the apoA-I isoforms in the lymph (d less than 1.006 g/ml lipoproteins), which may be of significance in their plasma catabolism.